Treatment outcomes of cutaneous leishmaniasis due to Leishmania aethiopica: A systematic review and meta-analysis

Background Leishmania aethiopica is a unique species that causes cutaneous leishmaniasis (CL), and studies evaluating treatment outcomes for this condition reported inconsistent findings. This study aimed to summarize the evidence on treatment outcomes of CL caused by L. aethiopica to support decisions or propose further study. Methods We searched PubMed, Scopus, and ScienceDirect. In addition, we searched grey literature on Google Scholar and performed manual searching on the reference list of articles. Two authors did the screening, selection, critical appraisal, and data extraction. With the narrative synthesis of evidence, we performed a random effects model meta-analysis using the metaprop package in Stata 17. We did sensitivity and subgroup analyses after assessing heterogeneity using the I-squared test and forest plots. The funnel plot and Egger’s test were used to assess publication bias. Results The review included 22 studies with 808 participants, and the meta-analysis included seven studies with 677 participants. Most studies documented treatment outcomes with antimonial monotherapy, and only one study reported outcomes with combination therapy. The overall pooled proportion of cure was 63% (95% CI: 38–86%). In the subgroup analysis, systemic antimonial monotherapy showed a cure rate of 61%, and the proportion of cure was 87% with topical therapy. Topical therapy showed a better cure for the localized clinical phenotype. A cohort study documented a cure rate of 94.8% with combination therapy for the localized, mucocutaneous, and diffuse clinical phenotypes. The pooled proportion of unfavourable outcomes was partial response (19%), relapse (17%), discontinuation (19%), and unresponsiveness (6%). Conclusions The pooled proportion of cure is low with antimonial monotherapy. Despite limited evidence, combination therapies are a promising treatment option for all clinical phenotypes of CL caused by L. aethiopica. Future high-quality randomized control trials are needed to identify effective monotherapies and evaluate the effectiveness of combination therapies.


Introduction
Cutaneous leishmaniasis is a category one emergent uncontrolled neglected tropical skin disease (skin NTD) caused by obligate intracellular protozoa; Leishmania [1].More than 20 Leishmania species cause CL across the globe [2].The unique species Leishmania aethiopica (L.aethiopica) is the predominant cause of endemic CL in the Ethiopian highlands and Mount Elgon area of Kenya [3].Also, a case of CL due to Leishmania tropica (L .tropica)reported from Awash valley, Ethiopia [4].The main clinical phenotypes of CL due to L. aethiopica are localized cutaneous leishmaniasis (LCL), mucocutaneous leishmaniasis (MCL) and diffuse cutaneous leishmaniasis (DCL) [5].While LCL is the commonest type, MCL is relatively common and DCL is a rare event [3].
Treatment outcomes vary depending on the clinical phenotype of CL and causative Leishmania species [1,5].In a meta-analysis of randomized control trials (RCTs), topical treatment with 15% paromomycin plus 12% methylbenzethonium chloride (PR-MBCL) was 56% less efficacious compared with photodynamic therapy for CL due to L. major.Thermotherapy showed three times more likely cure for CL due to L. tropica compared with intramuscular pentavalent sodium stibogluconate (IM-SSG V ) [6].Conventional drugs are becoming ineffective because of species level unresponsiveness [7].The symbiont Leishmania RNA virus 2 (LRV2) in L. major increase treatment failure in Uzbekistan [8].
In Ethiopia, the only randomized phase II trial conducted in Amhara region showed 69% reduction in LCL lesion size at 16 weeks, after a four-week topical application of Shiunko ointment compared to the placebo group [9].Observational studies evaluating CL treatment outcomes showed cure rates ranging from 12.5% to 85% for different clinical phenotypes of CL due to L. aethiopica [10][11][12][13].Unfavourable treatment outcomes, such as relapse, change in treatment regimen, treatment extension and failure also showed variation across observational studies in Ethiopia [11][12][13][14].In Kenya, three CL cases due L. aethiopica treated with high dose (18 to 20mg/kg twice daily) intravenous (IV) SSG V [15] for 30 days had shown disappearance of parasite from skin smear and culture after 14 to 27 days, clinical healing of lesion and no recurrence at 3 to 18 months follow-up [16].There are also studies which assessed treatment outcomes of CL due to L. aethiopica in immigrants.Complete lesion resolution was observed after 45 days of intramuscular meglumine antimonate treatment in Belgian traveller returned from northern Ethiopia [17].
Randomized control trial that assessed the effectiveness of different treatment options for CL due to L. aethiopica to base treatment decision hardly exist.Observational and very few nonrandomized clinical trials that reported treatment outcomes for CL due to L. aethiopica had inconsistent findings to base clinical decisions.Therefore, the aim of this systematic review and meta-analysis was to aggregate available evidence on the treatment outcomes of CL due to L.

Commented [ba11]:
This paragraph is not related to the different species of Leishmania.So, either remove it totally or make it short.

Commented [ba12]:
Here, please writes the guidelinerecommended treatment regimen in the country if any.

Commented [ba13]
: "Summarizes" is a better word than the aggregate aethiopica to support clinical decision-making and suggest potential treatment alternatives for future randomized control trial.

Reporting
The protocol of this systematic review was registered in the PROSPERO database [CRD42022306698].We used the Preferred Reporting Items for Systematic Review and Metaanalysis (PRISMA) checklist 2020 [18] to report the results (S1 Checklist).

Eligibility criteria
We included studies on human being that reported treatment outcomes of CL due to L. aethiopica, published in English but without restriction to year of publication and study design.To include all article that report treatment outcomes of CL due L. aethiopica everywhere, we did not limit studies by country.

Search strategy
We developed the Mnemonic PEO (Problem, Exposure, Outcome) to organize searching.A comprehensive searching of PubMed, Scopus and ScienceDirect databases was done using Boolean operators 'AND' and 'OR' as appropriate.In PubMed and Scopus, we used the search terms: "Leishmaniasis" OR "Oriental sore" OR "Cutaneous Leishmaniasis" OR "Diffuse Cutaneous Leishmaniasis" OR "Old World Cutaneous Leishmaniasis" OR "Mucocutaneous Leishmaniasis" OR "L.aethiopica" OR "Leishmania aethiopica" AND "Cryotherapy" OR "Heat therapy" OR "Thermotherapy" OR "Systemic therapy" OR "Localized therapy" OR "Oral therapy" OR "Miltefosine" OR "Sodium stibogluconate" OR "Antimoniate" OR "Glucantime" OR "Meglumine antimoniate" OR "Liquid nitrogen therapy" OR "Paromomycin" OR "Amphotericin b" OR "Withholding treatment" OR "Conservative treatment" OR "Laser therapy" AND "Treatment outcome*" OR "Treatment failure" OR "Treatment Cure" OR "Relapse" OR "Unresponsiveness" OR "Non-responsiveness" OR "Failure" OR "Cure" OR "Outcome" OR "Partial response" OR "Dropout" OR "Treatment extension" OR "Resistance".Additionally, the following key terms were used to search in ScienceDirect: "Localized Cutaneous Leishmaniasis" OR "Mucocutaneous Leishmaniasis" OR "Diffuse Cutaneous Leishmaniasis" OR "Old World Cutaneous Leishmaniasis" OR "Leishmania aethiopica" AND "Treatment relapse" OR "Treatment failure" OR "Treatment Cure" OR "Partial response".The detailed searching strategy for the major databases is available in the S1 Text.To access grey literature, we searched the first 20 hits in Google scholar using the review title.Moreover, manual searching of reference lists was done.

Study selection process and quality assessment
All studies identified from databases and grey literature search were imported into Endnote X8 to remove duplicates.Two independent reviewers (AYA and KA) did the screening and selection of studies based on the title and abstract.These two reviewers did the critical appraisal of full-text articles.The Newcastle Ottawa Scale (NOS) [19] and risk of bias assessment tool version 2 (RoB2) [20] were used to appraise observational and clinical trial studies, respectively.All differences throughout the process were resolved through discussion.We adapted and used the PRISMA 2020 flow diagram to present the study selection process [21].

Data extraction and items
We developed Microsoft excel sheet to extract the data.The excel was developed based on PRISMA template, and piloted before use.Two reviewers (AYA and KA) extracted the data.The following data items were extracted: 1) primary author and year of publication; 2) study design; 3) diagnosis confirmation method; 4) sample size; 5) clinical phenotype of CL (LCL, MCL and DCL); 6) treatment detail (route, dose, dosage and treatment type); 7) characteristics of participants; and 8) treatment outcomes.

Data analysis
We did narrative synthesis of evidence and meta-analysis.In addition to narrative synthesis of evidence, we did meta-analysis of proportions for treatment outcomes, ignoring heterogeneity [22].Meta-analysis was done using metaprop package in Stata 17.Since this package uses Freman-Tukey Transformation (ftt), the study specific and pooled confidence intervals (CI) were admissible and avoids exclusion of proportions close to 0% or 100% from the meta-analysis [23].
The pooled proportions of cure, partial response, relapse, dropout and unresponsiveness reported Commented [ba14]: Why do you ignore the heterogenicity?How do you do MA while there is significant heterogenicity?

Commented [ba15R14]:
by two or more studies were estimated using random effects model and presented in forest plot [24].
To assess heterogeneity, I-squared statistics <75% and overlap confidence intervals on the forest plot were used [25].We did subgroup analysis and sensitivity analysis to address high heterogeneity.While sensitivity analysis was presented graphically, we did subgroup analysis by study design, clinical phenotype of CL and type of treatment.Moreover, we assessed small study effect subjectively using funnel plot, and objectively using Egger's test.

Characteristics and quality of included studies
A total of 3520 studies were identified from all sources.We retrieved 52 studies pertinent to our objective.Of the 52 studies, 33 excluded, because of reasons mentioned in the PRISMA flow diagram (Fig 1).Then, 22 studies reporting treatment outcomes of CL due to L. aethiopica were included in the systematic review, of which seven studies were used in the meta-analysis.
Commented [ba16]: See above comment, you already plan to ignore heterogenicity, what is the importance of this sentence and subsequent below ?

Treatment outcomes
We classified treatment outcomes as positive if it favors cure; otherwise, we classified it as negative.Positive outcomes reported in the studies include cured, improved, negative/decrease in parasite density, good response, reduction in the size of the lesion, or resolution of the lesion without scar.On the other hand, negative outcomes were recurrence, resistance, treatment extension, partial response, relapse, dropout and unresponsiveness, progression to more severe stage and toxicity.Furthermore, we synthesized predictors of treatment outcomes.

Positive outcomes
In Ethiopia, as shown in Table A in S2 Table, six cases with concomitant lesion (CL and leprosy) treated with systemic pentamidine responded well, but the authors did not report the route, dose and duration of treatment, and nothing documented how outcome was ascertained [27].Two prospective cohort studies also report positive treatment outcomes [10,13] IM-Meglumine antimonate (MA) resistant cases treated with 4m/kg IM pentamidine every-otherday (QoD) for 20 days had shown clinical and microscopy cure at six months [10].After 100-150 mg oral miltefosine daily treatment, good clinical improvement reported in 70%, 28% and 14.3% of cases at day 28, day 90 and day 180 follow-up, respectively [13].In a prospective follow-up study, 14% of LCL patients treated with six doses of IL-SSG V showed good response at day 90 [40].As shown in

Commented [ba18]:
Commented [ba19]: Good if you rephrase it like this "The most common form of treatment described across studies was antimonial alone, but eight studies report combination therapy".
Treatment outcomes were also reported among travelers and immigrant cases [17,32,36].A Belgian traveller who returned from Ethiopia treated with 1.5 mg/5ml IM-MA for 45 days showed clinical resolution at 45 days and no relapse at six months [17].An immigrant from Eretria to Germany treated with 200 mg/day IV-amphotericin B for 22 days healed at 12 months of followup without recurrence [36].Three Ethiopian migrants in Israel had shown parasite clearance (smear and culture negative), clinical improvement and complete healing without scar at 10 days, 20 days, and 40 days after treatment, respectively, with 15% paromomycin sulfate and 12% methyl benzethonium chloride combination ointment [32].

Three clinical trials done in Ethiopia reported positive treatment outcomes (Table C in S2 Table).
A phase II clinical trial compared the efficacy of four weeks twice daily Shiunko ointment with placebo among 20 LCL cases in each group.Five cases in the Shiunko group and four in the placebo group were cured.Six cases responded partially in both groups.Additionally, the Shiunko group showed 69% reduction in lesion size but only 22% in placebo group at 16 weeks [9].Among twelve cases (six in each group), a daily dose of three combination (isoniazid 300 mg, amithiozone 150 mg, and rifampicin 600 mg) drugs for eight weeks was compared with fifteen doses of 4mg/kg IM QoD pentamidine.In the combination group, one case improved without inflammation and scar at 12 weeks, and six cases in pentamidine group had negative smear at four weeks.In this trial, the clinical phenotype of CL was not reported [29].Another clinical trial compared 200 mg itraconazole daily dose with placebo in 14 patients (four DCL and 10 LCL cases), seven in each group.No difference between the two group after four weeks [33].
Four case series studies [26,30,31,34] documented positive treatment outcomes among DCL cases.Among 33 cases, cure was recorded in seven with pentamidine, three with chloroquine, one with primaquine and one with Amphotericin B (Table A in S2 Table ).Yet, the dates of outcome measure were not specified [26].In other study, two DCL cases treated with 4 mg/kg IM pentamidine daily for 2 weeks showed decrease in parasite number, but the date of outcome measure was not reported.The test of cure was done using smear microscopy and biopsy [30].
Similarly, three DCL cases treated with 1g 25% Chlorpromazine and 25% 50g Vaseline ointment for one month showed disappearance of clinical signs of inflammation in one case and decreased lesion size in another [31].Two cases treated with 14 mg/kg IM-aminosidine daily for 60 days and one case treated with a combination of the same dose of Aminosidine plus 10 mg/kg IM-SSG daily.All cases got cure after two months of extended treatment [34].

Negative outcomes
In Ethiopia, two CL-HIV co-infected cases treated with 20 mg/kg/day IV-SSG V for 30 days had recurrence at five months of follow-up [35].A case of MCL progressed to DCL after 30 days treatment with a combination of 20 mg/kg/day IM-SSG V and 15 mg/kg/day paromomycin, and IM-SSG V alone for extra 60 days [38].No change to lesion observed in five MCL cases treated with four to eight weeks 500 mg oral metronidazole, and two cases report burning sensation [28].
A series of 33 DCL patients were unresponsive and had experienced toxicity for various treatment options (Table A in S2 Table ).In this series, more than half of the cases were unresponsive to pentamidine (n=24) and pentostam (n=17).High toxicity (3+) was recorded in pentamidine and amphotericin B treatment [26].While five LCL cases got worse of their lesion, two cases were unresponsive to IL-SSG V in a cohort study [40].).In a phase II clinical trial that compared Shiunko ointment versus placebo, six, eight and four of the twenty LCL cases showed partial response, treatment failure and dropout, respectively [9].Similarly, after four weeks treatment with 50 mg oral itraconazole versus placebo, five of the seven cases had active CL lesion at follow-up visit [33].In a study that compared a combination of 300 mg isoniazid, 150 mg amithiozone and 600 mg rifampicin versus pentamidine five of the six cases showed parasitological or clinical improvement after eight weeks of daily treatment [29].

Predictors of outcome
This study showed that predictors of treatment outcomes were investigated exhaustively.Only two cohort studies in Ethiopia report predictors of CL treatment outcome [13,40].Among LCL cases treated with IL-SSG V , high probability of cure at day 90 was associated with older age, SSS grade less than +6 and being male [40].In a cohort study that assessed outcome of patients treated with 150mg/day oral miltefosine for one month, MCL predicted low chance of relapse at day 180 compared to LCL [13].Large lesion size decreases the chance of cure at day 90 in both studies [13,40].

Meta-analysis
Seven studies [10-13, 37, 39, 40], all from Ethiopia, were included in the meta-analysis to estimate the pooled proportions of treatment outcomes of CL due to L. aethiopica.All studies included in this meta-analysis scored five and above with the NOS.Though the subjective judgment of funnel plot showed asymmetry (Fig 2), the insignificant statistical test with Egger's test (p-value= 0.329) indicated no publication.We estimated the pooled proportions of various treatment outcomes based on the meta-analysis of observational studies.However, high quality clinical trials that evaluate the effectiveness of available treatment options are lacking.
Moreover, the proportion of cure among DCL cases estimated from four studies was 0.51 (95% CI: 0.23-0.79;I 2 =71.74%) (Fig 6).The cure rate of DCL cases with monotherapy (parenteral SSG V or oral miltefosine) was below 50% [12,13,39].In one of the studies [37], 80% DCL cases had shown cure with a combination of IM-SSG V and Allopurinol, 86% with a combination of IM-Commented [ba23]: In Figure 5, the is only one study in the subgroup analysis (Seife T/2018).However, there must be at least two studies in each subgroup.The finding of this analysis skewed your conclusion and recommendation.This must be corrected and revise you conclusion accordingly.SSG V , Cryotherapy and IL-SSG, and the overall cure rate 83%.The cryotherapy and IL-SSG v was used to treat satellite lesion.

Sensitivity analysis
In the sensitivity analysis no study affected the overall pooled proportion of cure, but a study by van Henten S. et al [13] showed some level of influence on the overall cure rate (Fig 7).

Proportions of negative outcomes
In a cohort study, negative outcome (unspecified) was 15% with four weeks 20mg/kg/day IL-MA every 3 days treatment [10].This study reported 28% resistance with four weeks 20 mg/kg/day IV-MA treatment.In addition, 52% of relapsed cases retreated with the same does of IV-MA for four weeks were not cured [10].van Henten S. et al [13] reported 32.3% relapse at day 180 with four week 50 mg daily oral miltefosine treatment.Furthermore partial response was 20.4% [13] and 25% [12].The detail on treatment outcomes at different time points is presented in Table B in The pooled proportions of negative outcomes (partial response, relapse, dropout and unresponsiveness) reported in two or more studies were estimated (Table 1).All I-square tests showed low (<75%) heterogeneity in the pooled estimate of negative outcomes.

Discussion
We noted that systemic antimonial alone or in combination, local (topical) and combination therapies were used to treat CL case due to L. aethiopica.Less than two-third of the cases cured with these treatment options.This finding also showed that negative treatment outcomes, such as partial response, relapse, dropout and unresponsiveness were significant.The studies we reviewed documented treatment outcomes of CL due to L. aethiopica with a range of treatment options.For example, a case series reported outcomes with more than ten treatment options [26].This might indicate unavailability of standardized treatment guideline that contributed to selection of various treatment options relying on individual expertise and experiences of the clinicians.The national leishmaniasis diagnosis and treatment guideline of Ethiopia underpinned the use of unstandardized and non-evaluated treatment options [41].Thus, the evaluation of available treatment options and standardized treatment of CL due to L. aethiopica is a national priority.
In this study, the overall pooled proportion of cure was 63% with available treatment options.This proportion was low with systemic antimonial monotherapy such as IV/IM-SSG V and high with local and combination therapies.High proportion of cure with local therapy was limited to LCL, but combination therapy works across all clinical phenotypes.The proportion was as low as 29% with systemic antimonies alone.Low cure rate might be due to low efficacy of antimonial drugs for CL due to L. aethiopica, and high drug resistance of this species.As such, the proportion of cure was only 61% with systemic antimonial monotherapy.In our review, resistance to systemic meglumine antimonate was 28% [10].A systematic review also report about increasing resistance of Leishmania species for SSG V drug [5].In our review, SSG V is the mainstay of treatment for CL due to L. aethiopica yet the cure outcome documented is poor.This implies the necessity of future study assessing the resistance pattern of this protozoa for existing treatment options.
In the subgroup analysis, CL due to L. aethiopica had shown 87% cure rate with local therapy.
This finding was in line with a systematic review [42] that report greater than 80% cure rate for CL due L. major and L. tropica with topical agents.Thermotherapy, paromomycin and combinations, CO2 laser, 5-aminolevulinic acid hydrochloride (10%) plus visible red light (633 nm) and cryotherapy were used [42].However, only liquid nitrogen-based cryotherapy and IL-SSG were used in our review [11,37,39,40].In all the studies were reviewed, IL-SSG was the main topical agent.The effectiveness of this topical agent alone was low.For example, in a cohort study we reviewed, the cure rate was 60% with IL-SSG [40].Liquid nitrogen-based cryotherapy is expensive with an estimated cost of 4 USD per patient [11].So, other topical agents such as thermotherapy, CO2 laser and thermotherapy could be considered for CL due to L. aethiopica.
In a cohort study we reviewed, combination of SSG V with other treatment options showed high cure rate ranging from 78.6-96% [37].This proportion varied across the clinical phenotypes.With combinations of IL-SSG V and cryotherapy, the proportion was 96% in LCL cases.In MCL cases, the proportion was 79% with a combination of IM-SSG V and IL-SSG V , and 85.7% with a combination of IM-SSG V and Allopurinol.In DCL cases, the proportion of cure was 80% with a combination of IM-SSG V and Allopurinol, and 86% with a combination of IM-SSG V , cryotherapy and IL-SSG V [37].A systematic review also report 70-100% cure rate with a combination of pentavalent antimonial with other alternatives [43], but the species of leishmania and the clinical phenotypes were not reported.Thus, clinicians could consider combination therapies to increase cure rate of CL due to L. aethiopica, and combination therapy should be the option included in the national leishmaniasis diagnosis and treatment guideline.
The pooled proportion of unfavorable outcomes of CL due to L. aethiopica were partial response (19%), relapse (17%), dropout (19%) and unresponsiveness (6%).The proportion of relapse in our finding was higher than 2-6% relapse reported in systematic review that assessed effectiveness of different topical agents for LCL cases due to L. major and L. tropica [42].High proportion of relapse in our finding might be due to severe clinical phenotypes such as MCL and DCL associated with L. aethiopica [3].These severe clinical phenotypes are difficult to treat [44].Similarly, partial response and unresponsiveness were unacceptably high in our finding.This might be due to high resistance of L. aethiopica for available treatment options; as described earlier.The dropout might also increase the probability of partial response, relapse and unresponsiveness.Hence, due account of drug resistance and reducing dropout should not be understated to counter negative outcomes.
In general, this systematic review and meta-analysis provided an updated evidence on treatment outcomes of CL due to L. aethiopica.However, the study was not without limitations.Though we used the random effects model and subgroup analysis, heterogeneity persisted.Hence, the pooled estimates might be affected by high heterogeneity between studies, but it does not mean that the pooled estimates are not useful for decision.Besides, the number of studies in the meta-analysis were below ten which might negatively affect the reliability of the funnel plot and Egger's test.
Yet, it is possible to pool the effect size from two studies, and evidence from two studies is more powerful for decision than a single study evidence.Furthermore, clinical trial studies included in this study had low quality, involve small sample size, and none of the studies assessed the effectiveness of either local, combination or systemic antimonial therapy using high quality randomized control trial.

Conclusions
The pooled proportion of cure for CL due to L. aethiopica was low.While the cure rate was much lower with systemic antimonial monotherapy, it was better with local and combination therapies.
The LCL cases had shown higher cure rate with combination and local therapies; whereas, the MCL and DCL cases had better cure with combination therapy.Additionally, unfavourable treatment outcomes such as partial response, relapse, dropout and unresponsiveness were significant.The effectiveness of neither combination therapy nor other therapeutic options were evaluated using high quality randomized clinical trial.Therefore, combination therapies could be better candidate to increase treatment success across all clinical phenotypes, but we recommend further evaluation of the effectiveness of combination therapy using randomized control trials.Financial disclosure: The author(s) received no specific funding for this work.The Skin Health Africa Research Program project financially supported the first author in the form of student stipend, but the project did not decide in the design, analysis and reporting of this study.

Abbreviations
report combination therapy.The details of study designs, sample size, confirmation of diagnosis, treatment type, dose and dosage, patient characteristics, treatment outcomes were presented in S2 Table.

Fig 3 .
Fig 3. Forest plot of the pooled proportion of CL treatment cure

Fig 4 .
Fig 4. Subgroup analysis forest plot of treatment cure by study design

Fig 5 .
Fig 5. Subgroup analysis forest plot of treatment cure by treatment type As shown in Figure 6, we did subgroup analysis across clinical phenotype (LCL, MCL and DCL).
Author contributions: AYA: Conceptualization, data curation, formal analysis, methodology, software, writing-original draft and writing-review and editing; LD: Data curation, methodology, writing-review and editing; MK: Data curation, methodology, writing-review and editing; EG: Data curation, methodology, writing-review and editing; KA: Data curation, methodology, writing-review and editing Commented [ba24]: These sentences should be modified.In the presence of high heterogeneity, what is the importance of determining the pooled estimate ?Commented [ba25]: Please support with a reference.Commented [ba26]: Is there any recommendation to pool effect from a single study?see other comments too.Commented [ba27]: See the comments about figure 5.
Table A in S2 Table, five Kenyans treated with high (18-20mg/kg twice daily)